During meiosis, cells undergo one round of DNA replication followed by two rounds of cell division to form haploid gametes. In meiosis I, homologous chromosomes segregate while in meiosis II, the sister chromatids segregate before cytokinesis to form haploid cells. Topoisomerase 2 is an enzyme that helps to facilitate DNA disentanglements. This enzyme has been studied extensively in mitosis but not in meiosis. Previously, we demonstrated that a temperature sensitive mutant,
top-2(
it7), causes chromosomes to fail to segregate in spermatogenesis when incubated at 24 deg C. The
top-2(
it7) mutation changes an arginine at amino acid 828 to cysteine. This mutation lies within an ?/? fold of TOP-2 called the tower subdomain that is located within the catalytic domain, a region that has been proposed to interact with DNA. Structural modeling of the TOP-2 protein indicates that the wild-type arginine makes several interesting contacts with other amino acids, including a salt bridge with glutamate at amino acid site 960. We hypothesize that the arginine at position 828 is particularly important for the structure and function of the enzyme. We have characterized several amino acid substitutions at Arg828. We found that an Arg828Ala change has high embryonic viability at 15 deg C but significantly reduced viability when incubated at 25 deg C. This mutation has intermediate segregation defects at 25 deg C that are not as severe as those seen in
top-2(
it7). An Arg828Trp change results in sterility due to the absence of germline development. Characterization of an Arg828Lys change results in reduced viability when incubated at 24 deg C. Examination of Arg828Lys mutant germlines will reveal whether any chromosomal segregation defects are present. Future directions will include the examination of protein levels in the various
top-2 mutants and the characterization of additional amino acid substitutions.